Project Summary/Abstract Drug discovery depends crucially upon reliable assays for biological activity. Live cell assays provide a rich environment for measuring biological activity. Coupled with genetically encoded fluorescent biosensors, live cell assays have the potential to provide read-outs with unprecedented specificity for particular signaling pathways. Although widely used for basic research applications in living cells, genetically encoded fluorescent biosensors have had little impact on drug discovery because of difficulties in measuring and interpreting fluorescence intensity read-outs, including poor signal to noise ratios, variability in cell expression, and interference from fluorescence emitted by compounds. This Phase 1 project will demonstrate the feasibility of a new strategy that combines highly specific biosensors with extremely fast fluorescence lifetime measurements to produce the speed, sensitivity and specificity needed for high throughput screening applications. This approach employs an alternative fluorescence measurement based on fluorescence lifetime that is much faster than time-correlated single photon counting (TCSPC), yet also more precise. It operates in non-imaging mode which makes for simple data interpretation and minimizes background fluorescence. It goes far beyond the expected incremental improvements to image-based technologies. Our preliminary data demonstrates the tremendous potential for robust live cell assays when lifetime methodology is applied to measuring genetically encoded fluorescent sensors. Our specific aims will accomplish the vital proof of principle steps and set the direction for our long term objectives of producing a robust live cell drug discovery platform within 5 years. PUBLIC HEALTH RELEVANCE: New assays for biological activity are urgently needed to develop safe and effective drugs that provide better treatment outcomes and improved human health. This proposal addresses the technical challenges associated with using fluorescent live-cell assays and has strong potential to reduce the cost and improve the reliability of drug discovery processes.